Nucleic acids such as DNA, are used extensively in the field of molecular biology for research and clinical analyses. Common methods for analyzing DNA are Southern blotting, amplification through methods such as polymerase chain reaction (PCR), and sequencing. Using these methods, differences in DNA sequence are determined to aid in gene identification, population screening, pathogen identification and diagnostic testing. All of these analyses require purified DNA samples as the basis for consistent and valid results.
There are numerous nucleic acid purification methods that fall into two general categories, liquid phase and solid phase purification. In liquid phase purification, the DNA remains in the liquid phase while impurities are removed by precipitation and/or centrifugation. In solid phase purification, the DNA is bound to a solid support while impurities are selectively eluted. Both purification strategies utilize conventional methods, which require many steps and often hazardous reagents, as well as more rapid methods, which require fewer steps and usually less hazardous reagents.
Using conventional liquid phase methods, DNA is most commonly isolated using density gradient centrifugation, organic solvent extraction, or salt precipitation. Protocols describing these purification methods are given in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., 7.19-7.25, 9.16-9.19, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) and Ausubel, et al., Current Protocols in Molecular Biology, 4.4.2-4.4.4 (1987). Briefly stated, the liquid phase purification methods of density gradient centrifugation, phenol-chloroform extraction, and salt precipitation generally require four main steps: lysing the cells to release the DNA from cellular and nuclear membranes; removing impurities (such as proteins, lipids and carbohydrates); concentrating by alcohol precipitation; and then rehydrating the DNA in a hydration solution. The major differences among these three methods occur during the second step, where impurities are removed from the DNA by density differentiation, organic-aqueous phase partitioning, or selective salt precipitation.
A conventional liquid phase purification method for purifying blood dried on specimen collection cards (Guthrie cards) is described by McCabe et al., Human Genetics, 75, 213-216 (1987). The method follows closely a procedure for liquid blood described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., 9.16-9.19, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). In this phenol extraction method, dried white cells are removed from the collection paper by rehydrating with a saline solution. The white cells are incubated in a buffer to lyse the cells. Then, three phenol extractions are performed to remove protein impurities followed by three ether extractions to remove the phenol. The DNA is concentrated by sodium acetate-ethanol precipitation, washed with 70% ethanol and then rehydrated in a standard DNA hydration solution. Several reagents (ten), two of which are hazardous (phenol and ether), are typically required for this method. Although these conventional methods typically yield highly purified DNA preparations, they are laborious and hazardous.
As with liquid phase purification, conventional solid phase methods have been developed to generate highly purified DNA. Generally these methods require four general steps: lysing cells to release DNA from cellular and nuclear membranes; binding the released DNA to a solid support; washing away impurities; and then eluting the purified DNA. The first two steps, lysing the cells and binding the released DNA, usually require hazardous reagents at high concentration.
For solid phase DNA purification, many solid supports have been used including membrane filters, magnetic beads, metal oxides, and latex particles. Probably the most widely used solid supports are silica-based particles (see, e.g., U.S. Pat. No. 5,234,809 (Boom et al.); International Publication No. WO 95/01359 (Colpan et al.); U.S. Pat. No. 5,405,951 (Woodard); International Publication No. WO 95/02049 (Jones); WO 92/07863 (Qiagen GmbH). For example, the method disclosed in U.S. Pat. No. 5,234,809 (Boom et al.) uses a high concentration chaotropic solution to bind DNA to silica particles and requires six centrifugation steps and five reagents to purify DNA from whole blood. Disadvantages of this method are the use of a particulate suspension, the use of many centrifugation steps, and the use of hazardous reagents, such as guanidinium isothiocyanate and acetone.
One means for simplifying the conventional solid phase purification procedures is to eliminate the elution step and use the DNA while it is bound to a solid support for subsequent analyses, such as amplification. Thus, by using immobilized DNA, usually at least one reagent and one step is omitted. For example, U.S. Pat. No. 5,234,809 (Boom et al.) describes such a method for purifying DNA, although it is not present in a complex mixture such as blood. Using the method described above, but omitting the elution step, reduces the number of reagents and steps by one.
In another example, U.S. Pat. No. 5,496,562 (Burgoyne) describes a method of purifying cellulose filter paper containing dried blood that uses four reagents during four phenol washes and five isopropanol washes. After drying, a small piece of the filter paper is cut from the square and used directly as a substrate for PCR amplification. Despite the use of bound DNA for analysis, these methods still require many steps and hazardous reagents.
Recently, there has been a trend toward developing more rapid and simple methods for both liquid and solid phase purification. This has been driven in part by the development of DNA amplification assays which reduce the time necessary for analysis. As the number of DNA-based assays has increased in the field, there is a need for more rapid means of processing the biological samples. Also, using simpler methods reduces the risk of sample cross-contamination by reducing the number of sample handling steps. In addition, the simpler the method, the more readily the process may be automated.
One rapid liquid phase method for DNA purification uses a chelating resin to remove metal impurities from liquid blood or blood stains (Walsh et al., BioTechniques, 10, 506-513 (1991)). Using this method, blood cells are first washed with deionized water and then incubated with a suspension of the chelating resin and deionized water at 56° C. for 15-30 minutes. This incubation is followed by vortexing, incubating at 100° C. for 8 minutes, vortexing again, and removing the impurities by centrifugation. This method is rapid (completed in 45-75 minutes) and simple (requires only two reagents).
Another simple and rapid method for liquid phase DNA purification is described by Nordvag et al., BioTechniques, 12, 490-492 (1992). Starting with whole human blood, the blood cells are washed twice with a solution of 10 mM EDTA and 10 mM NaCl and collected by microcentrifugation after each wash. Then the cells are resuspended in 50 mM Tris(hydroxymethyl)aminomethanehydrochloric acid (Tris-HCl) buffer (pH 8.0) and boiled for 3 minutes prior to PCR amplification. For this purification method, only two reagents are required, both of which are generally nonhazardous. Furthermore, the method requires only approximately 15 minutes.
An even simpler single reagent method is described by Carducci et al., BioTechniques, 13, 735-737 (1992). Using this procedure, a 3 mm diameter blood spot is autoclaved for 3 minutes and then boiled for 5 minutes or sonicated for 10 minutes in a PCR-compatible buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 3 mM MgCl2 and 0.001 gelatin). The impurities remain bound to the disk following autoclaving while the DNA is recovered in the buffer.
All three of these liquid phase methods for DNA purification use low concentrations, nonhazardous reagents, and simplified methods. However, these three methods (Walsh et al., Nordvag et al., and Carducci, et al.) could be simplified further by eliminating the use of resins (which must be uniformly suspended prior to use), eliminating the repetitive washing of cells, or eliminating the cumbersome autoclaving of blood spots.
Rapid and simple methods for solid phase DNA purification have also been developed. The procedure of Berlin et al., Human Mutation, 1, 260-261 (1992) describes washing dried blood spots successively with a nonionic detergent-containing buffer. To elute the DNA from the filter paper, each sample is incubated at 65° C. for one hour with another nonionic detergent-containing buffer containing a proteinase K solution. A final incubation at greater than 95° C. for 10 minutes is necessary to inactivate the proteinase K. This method reduces the number of reagents required to three, but has the disadvantages of using an enzyme, a long incubation time, and a high incubation temperature (i.e., greater than 95° C.).
A rapid method using a membrane filter as a means of trapping DNA is disclosed in U.S. Pat. No. 5,234,824 (Mullis). Typically, this method requires a high concentration lysing reagent to lyse cells present in whole blood. Then, the lysate is applied to a filter and washed successively with a second lysing reagent and then either buffer or water to further purify the DNA. The DNA is eluted from the membrane by boiling for 15 minutes in water or in a buffered reagent containing magnesium chloride. Disadvantages of this solid phase purification method include the absence of a chelating agent in the purification and elution reagents, which can increase the probability of DNA damage (e.g., due to nucleases). In addition, there is a requirement for a cumbersome high temperature incubation (i.e., about 100° C.).
In another example, W.O. Pat. No. 96/18731 (Deggerdal) describes a method of purifying DNA from cells by mixing the cells with a detergent and a solid phase made up of magnetic beads. In this method, cells may be pre-lysed by the detergent to release the DNA which is subsequently bound to the solid phase. Alternatively, detergent may be added to a suspension made up of the cells and the solid phase, or the cells, detergent, and solid phase may be suspended together to allow the detergent to lyse the cells in the liquid phase and subsequently bind the DNA to the solid phase. However, this method involves multiple steps of adding or removing the liquid phase (i.e., detergent or the cell debris-detergent suspension) from the solid phase.
A very simple method is presented by Makowski et al., Nucleic Acids Research, 23, 3788-3789 (1995) in which 3 mm diameter disks punched from blood samples dried on cellulose collection paper are washed with deionized water (two 30 minute washes) and used directly for PCR amplification. As discussed in the above analysis of the '824 patent, a major disadvantage of using deionized water as a purification reagent is that the absence of a chelating agent increases the probability of DNA damage (e.g., due to nucleases). In addition, the absence of a detergent reduces the efficiency with which impurities are solubilized.
Nucleic Acids may be detected and quantitated by several means. Commonly, UV absorbance at a wavelength of 260 nm is used. A wavelength of 320 nm is used to determine background absorbance. Also, fluorimetry in the presence of Hoechst 33258 dye (e.g., Hoefer, DyNA Quant Fluorimeter, Pharmacia Biotech, Piscataway, N.J.), antibody detection strips (DNA Dipstick, Invitrogen, Carlsbad, Calif.), branched signal amplification (Chiron Corporation, Emeryville, Calif.), and quantitative PCR amplification (e.g., Applied Biosystems 7700, Perkin Elmer Applied Biosystems Division, Foster City, Calif.) are used to detect and quantitate nucleic acids.
As generally known and practiced, the purity of the DNA may be ascertained by measuring the absorbance at various wavelengths. The presence of impurities such as proteins, lipids, carbohydrates, cellular debris, etc. can increase the measured absorbance. In contrast, pure nucleic acids, especially DNA used in PCR amplification, have a substantially lower absorbance at established wavelengths.
Currently, there are numerous nucleic acid amplification systems available. While the most commonly used amplification methods are Polymerase Chain Reaction (PCR), other target amplification technologies include Ligase Chain Reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA), Self-sustained Sequence Replication (SSR or 3SR), Strand Displacement Amplification (SDA), and Transcription Mediated Amplification (TMA).
PCR is used routinely to amplify one or more targeted nucleic acid sequences within a sample or mixture of nucleic acids. This process is disclosed in U.S. Pat. No. 4,965,188 (Mullis). For each target nucleic acid sequence to be amplified in this process, separate complementary strands of nucleic acid are treated with two primers selected to be substantially complementary to portions of the target nucleic acid within the two strands. A thermostable enzyme (a polymerase) is generally used to extend the primers to form complementary primer extension products. When these are separated into their complementary strands, they serve as templates to extend the complementary primer into the target nucleic acid sequence. When separated, these in turn act as templates for synthesis of additional nucleic acid sequences. The PCR amplification process involves a series of simple steps. These include temperature cycling to cause hybridization of primers and templates, polymerase mediated synthesis of the primer extension products, and separation and subsequent annealing of the strands of template strands and the synthesized target nucleic acid sequences. Thus, there is an exponential increase in the amount of targeted nucleic acid sequences synthesized. PCR amplification is a very sensitive process. Therefore, a very high purity of starting sample is necessary.
LCR is another diagnostic technique that is often utilized in conjunction with a primary PCR amplification. LCR employs a thermostable ligase and allows the discrimination of DNA sequences differing in only a single base pair. LCR depends on highly pure NA templates due to its sensitivity.
Purified nucleic acids can be further analyzed by Southern hybridization, or Southern blotting as it is more commonly known. Southern blotting is the capillary transfer of DNA fragments from gels to various types of filter paper. It allows the researcher to detect rare sequences in a complex population of restriction fragments and is useful in gene cloning, reverse genetics, and the analysis of restriction-fragment-length-polymorphisms (RFLP's) for human genetic disease diagnosis. Southern blotting involves the digestion of DNA with one or more restriction enzymes, followed by a size separation by electrophoresis on an agarose gel. The DNA is then denatured in situ and transferred from the gel to a membrane (e.g., nitrocellulose or nylon). The DNA attached to the membrane is then hybridized to radiolabelled DNA or RNA, and autoradiography is used to locate the positions of bands complementary to the probe. Southern blotting is highly sensitive. A sequence of 1000 base pairs (bp) that occurs only once in the mammalian genome (i.e., 1 part in 3 million) can be detected in an overnight exposure if 10 μg of genomic DNA is transferred to the filter and hybridized to a probe several hundred nucleotides in length.
To advance the field of DNA sample preparation there is a need for solid phase DNA purification strategies. There is also a need for reagents and methods that are adaptable to solid phase purification strategies are not only simple and rapid but general in scope to maximize adaptability for automation. There is a need for reagents that are of generally low concentration, stable at room temperature (i.e., 20-25° C.), less hazardous (i.e., less corrosive, flammable or toxic), nonparticulate to eliminate the need for mixing, and protective of DNA quality. There is also a need for methods with few steps that can be performed using a variety of biological starting materials, whether hydrated or dried, especially as applied to routine testing as found in clinical laboratories. The reagents must not inhibit subsequent DNA analysis procedures by interfering with the buffering capacity of PCR buffers, or cause degradation of polymerase, primers or oligonucleotides used in DNA amplification. There is also a need for methods with few steps that can be performed using a variety of biological starting materials, whether hydrated or dried, especially as applied to routine testing as found in clinical laboratories.
The reagents and methods used in the solid phase purification strategy must also not interfere with standard methods for nucleic acid quantification, restriction enzyme digestion, DNA sequencing, hybridization technologies, such as Southern Blotting, etc., and amplification methods such as Polymerase Chain Reaction (PCR), include Ligase Chain Reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA), Self-sustained Sequence Replication (SSR or 3SR), Strand Displacement Amplification (SDA), and Transcription Mediated Amplification (TMA), or other DNA analysis.